Method Development of Efficient Protein Extraction in Bone Tissue for Proteome Analysis

Exploring bone proteome is an important and challenging task for understanding the mechanisms of
physiological/pathological process of bone tissue. However, classical methods of protein extraction
for soft tissues and cells are not applicable for bone tissue. Therefore, method development of efficient
protein extraction is critical for bone proteome analysis. We found in this study that the protein extraction
efficiency was improved significantly when bone tissue was demineralized by hydrochloric acid (HCl).
A sequential protein extraction method was developed for large-scale proteome analysis of bone tissue.
The bone tissue was first demineralized by HCl solution and then extracted using three different lysis
buffers. As large amounts of acid soluble proteins also presented in the HCl solution, besides collection
of proteins in the extracted lysis buffers, the proteins in the demineralized HCl solution were also
collected for proteome analysis. Automated 2D-LC−MS/MS analysis of the collected protein fractions
resulted in the identification of 6202 unique peptides which matched 2479 unique proteins. The identified
proteins revealed a broad diversity in the protein identity and function. More than 40 bone-specific
proteins and 15 potential protein biomarkers previously reported were observed in this study. It was
demonstrated that the developed extraction method of proteins in bone tissue, which was also the
first large-scale proteomic study of bone, was very efficient for comprehensive analysis of bone
proteome and might be helpful for clarifying the mechanisms of bone diseases.
Keywords: protein extraction • bone proteome • shotgun proteomics • tandem mass spectrometry • bone diseases
• biomarker discovery